Activation of Transcription Factor EB Is Associated With Adipose Tissue Lipolysis in Dairy Cows With Subclinical Ketosis

Abstract

Excessive lipid mobilization for adipose tissue caused by severe negative energy balance is the pathological basis for subclinical ketosis (SCK) in dairy cows. In non-ruminants, transcription factor EB (TFEB) was reported to play a role in the regulation of lipid catabolism, but its role in the control of lipolysis in the bovine is unknown. The present study aimed to determine whether the enhanced TFEB transcriptional activity contributes to lipolysis of adipose tissue in SCK cows, and to explore the possibility of establishing a therapeutic strategy by using TFEB as a target to control lipolysis. Thirty cows with similar lactation number (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9) were selected into a healthy control (n = 15) and SCK (n = 15) group, and used for subcutaneous adipose tissue biopsies and blood sampling. Adipocytes from healthy Holstein calves were used as a model for in vitro studies involving treatment with 10 μM isoproterenol (ISO) for 0, 1, 2 and 3 h, 250 nM of the TFEB activator Torin1 for 3 h, or used for transfection with TFEB small interfering RNA for 48 h followed by treatment with 10 μM ISO for 3 h. Compared with healthy cows, adipose tissue in SCK cows showed increased lipolysis accompanied by enhanced TFEB transcriptional activity. In vitro, ISO and Torin1 treatment increased lipolysis and enhanced TFEB transcriptional activity in calf adipocytes. However, knockdown of TFEB attenuated ISO-induced lipolysis in adipocytes. Overall, these findings indicated that enhanced transcriptional activity of TFEB may contribute to lipolysis of adipose tissue in dairy cows with SCK. The regulation of TFEB activity may be an effective therapeutic strategy for controlling overt lipolysis in ketotic cows.

Keywords: adipocytes; autophagy-lysosomal pathway; isoproterenol; lipolysis; transcription factor EB.

Figure 1

Transcriptional activity of transcription factor EB (TFEB) in white adipose tissue (WAT). (A) Western blot analysis of TFEB in the nucleus and cytoplasm of WAT of control cows (n = 15) and dairy cows with subclinical ketosis (SCK; n = 15). Representative blots are shown. (B,C) Quantification of protein abundance of nuclear and cytoplasmic TFEB, respectively. (D) Western blot analysis of phosphorylated TFEB (p-TFEB) and total TFEB in WAT of control cows (n = 15) and dairy cows with SCK (n = 15). Representative blots are shown. (E,F) Quantification of ratio of p-TFEB/TFEB and protein abundance of TFEB, respectively. (G,H) Relative mRNA abundance of TFEB and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) in WAT of control cows (n = 15) and dairy cows with SCK (n = 15). Data were analyzed using unpaired t-tests and expressed as mean ± SEM.

Figure 2

Isoproterenol (ISO) and Torin1 treatment activate transcription factor EB (TFEB) in calf adipocytes. Calf adipocytes were stimulated with 10 μM ISO for 0, 1, 2, or 3 h, or treated with 250 nM Torin1 for 3 h. (A) Western blot analysis of TFEB in the nucleus and cytoplasm of calf adipocytes. Representative blots are shown. (B,C) Quantification of protein abundance of nuclear and cytoplasmic TFEB, respectively. (D) Western blot analysis of phosphorylated TFEB (p-TFEB) and total TFEB in calf adipocytes. Representative blots are shown. (E,F) Quantification of ratio of p-TFEB/TFEB and protein abundance of TFEB, respectively. (G,H) Relative mRNA abundance of TFEB and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) in calf adipocytes. Data were analyzed using one-way ANOVA with Tukey's tests for data meeting homogeneity of variance or with Tamhane's T2 analysis for data of heteroscedasticity and expressed as mean ± SEM. The same letter (a-e) indicates no significant difference (P > 0.05), whereas different letters indicate a significant difference (P < 0.05).

Figure 3

Isoproterenol (ISO) and Torin1 treatment induce lipolysis in calf adipocytes. Calf adipocytes were stimulated with 10 μM ISO for 0, 1, 2, or 3 h, or treated with 250 nM Torin1 for 3 h. (A) Western blot analysis of phosphorylated hormone sensitive lipase (p-HSL), HSL and adipose triacylglycerol lipase (ATGL) in calf adipocytes. Representative blots are shown. (B,C) Quantification of ratio of p-HSL/HSL and protein abundance of ATGL, respectively. (D) Relative mRNA abundance of ATGL in calf adipocytes. (E) The content of glycerol (GC) in the supernatant of calf adipocytes. (F) The triglyceride (TG) content in calf adipocytes. (G) Oil Red O staining. Scale bar = 20 μm. Data were analyzed using one-way ANOVA with Tukey's tests for data meeting homogeneity of variance or with Tamhane's T2 analysis for data of heteroscedasticity and expressed as mean ± SEM. The same letter (a-e) indicates no significant difference (P > 0.05), whereas different letters indicate a significant difference (P < 0.05).

Figure 4

Knockdown of transcription factor EB (TFEB) reduces isoproterenol (ISO)-induced lipolysis in calf adipocytes. The cells were divided into 4 groups as followed: an siControl group [transfected with negative control of small interfering RNA (siRNA) for 48 h], and an siTFEB group (transfected with siRNA for TFEB for 48 h), an siControl + ISO group (transfected with negative control of siRNA for 48 h and then treated with 10 μM ISO for 3 h), and an siTFEB + ISO group (transfected with siRNA for TFEB for 48 h and then treated with 10 μM ISO for 3 h). (A) Western blot analysis of TFEB, phosphorylated hormone sensitive lipase (p-HSL), HSL and adipose triacylglycerol lipase (ATGL) in bovine adipocytes. Representative blots are shown. (B–D) Quantification of protein abundance of TFEB, ratio of p-HSL/HSL and protein abundance of ATGL, respectively. (E) Relative mRNA abundance of ATGL in calf adipocytes. (F) The content of glycerol (GC) in the supernatant of calf adipocytes. (G) The triglyceride (TG) content in calf adipocytes. (H) Oil Red O staining. Scale bar = 20 μm. Data were analyzed using one-way or two-way ANOVA with Tukey's tests for data meeting homogeneity of variance or with Tamhane's T2 analysis for data of heteroscedasticity and expressed as mean ± SEM. The same letter (a-c) indicates no significant difference (P > 0.05), whereas different letters indicate a significant difference (P < 0.05).